Glial cells (astrocytes, oligodendrocytes, microgliocytes) are the primary or secondary target of pathological processes in various diseases of the nervous system, in particular, in man, in leukoencephalitis, leukodystrophies, some forms of encephalopathy, some neurodegenerative diseases such as amyotrophic lateral sclerosis where there is a concomitant astrocytic gliosis with neuronal involvement, and lastly inflammatory diseases such as multiple sclerosis or Schilder's disease.
Multiple sclerosis (MS) is a chronic disease of the central nervous system in man, developing in a succession of phases of remission and exacerbation or according to a steady progression, and the anatomopathological feature of which consists of the formation of well-delimited patches of demyelination in the white matter of the brain and spinal cord (1). At histological level, these patches display, at the early stage of the lesion process, a degradation of the periaxonal myelin associated with an involvement of the glial cells responsible for this myelination, the oligodendrocytes (2). An inflammatory macrophage activation involving microglial cells (tissue macrophages resident in the central nervous system) as well as, probably, macrophages originating from infiltrated blood monocytes, is associated with this demyelination process and contributes to the destruction of the myelinated layers (3). In the center of the demyelinated patch, a relative depletion of glial cells is to be found, whereas a proliferation of astrocytes, or astrocytic gliosis, develops at the periphery and can, at a later stage, invade the demyelinated plaque to generate a fibrous or gliotic plaque, as is found on the site of old lesions (1). These sclerotic structures are the origin of the name given to the disease, multiple "sclerosis" (4).
Another feature of these plaques is their almost invariable association with a vascular element around which they appear to develop (5, 1). At histological level an adverse change in the blood-brain barrier (BBB) consisting of capillary endothelium is commonly observed in them (6). In effect, the vascular endothelium of capillary structures is normally butt-jointed in the central nervous system, except for the fenestrated capillaries associated with the choroid plexus, periventricular structures providing for the production of cerebrospinal fluid (7). This change in the BBB, marked by a parting of the endothelial cells and by the uncontrolled passage of a flow of plasmatic fluid and of cells of blood origin into the neuroglial parenchyma, may be visualized in "active" plaques by the magnetic resonance imaging (MRI) technique combined with the intravenous injection, into the patient under examination, of a solution of gadolinium. This compound enables a contrasted magnetic resonance signal to be obtained in the plasmatic fluid, and makes it possible to detect the abnormal passage of plasma into the nervous parenchyma with the edema resulting therefrom. It has been possible to show a correlation between the lesion-inducing activity of the plaques and the presence of this edema at the same level, as well as between the appearance and resorption of this edema and the regression of clinical exacerbations of the disease (8).
One of the decisive factors in maintaining a butt-jointed structure of the cerebral capillary endothelium, and hence in the endothelium of the BBB, consists of the underlying presence of cytoplasmic processes in the astrocytes, known as astrocyte feet (6). Probably, these astrocyte feet induce the formation or permit the maintenance of leakproof junction structures (of the zonula occludens type) which provide for the cohesion of the capillary endothelium barrier which is the material expression of the BBB. Now, various pathological models report the adverse change in the BBB associated with a depletion of astrocyte feet (9, 10).
Moreover, in the lesion process of MS, the adverse change in the BBB contributes to an amplification of the associated inflammatory response, through the afflux, rendered "free", of lymphoid cells originating from the blood circulation (11).
The contribution of the inflammation associated with immune cells is considerable in MS and participates in the lesion process, in particular via lymphocytes and self-reacting antibodies (12, 13). However, contrary to the autoimmune animal model of experimental allergic encephalitis, where the involvement of the BBB and the invasion of the nervous parenchyma by lymphoid cells of the circulating blood initiate the neuroglial lesion process (14), it is observed in MS that early lesion processes associated with a local macrophage reaction in the nervous parenchyma seem to precede the invasion of the tissue by lymphoid cells of the circulating blood (15, 3). It is thus apparent that lymphoid cells, and more especially lymphocytes, are not the prerogative of recent plaques (16). Furthermore, the density of the lymphocytic infiltration is most especially pronounced in the perivascular areas and at the periphery of the active plaques of demyelination (11, 3).
The initial stimulus at the origin of MS is at the heart of the debate about the etiology of MS (17). Arguments have been put forward, in turn, in favor of a viral (18), bacterial (19, 20), autoimmune (21, 13), toxic (22) or genetic (23, 24) hypothesis. In fact, it appears that a combination of a genetic predisposition to the action of a primary pathogenic agent may lead up to a devastating inflammatory and autoimmune process (25).
Different sequences of events may thus explain the demyelination and the functional neurological involvement in MS, without it having been possible to date to identify a decisive factor which might initiate and give a coherent explanation to the multitude of fragmented data which have accumulated concerning this disease.
Some molecules of bacterial or viral, or even endogenous retroviral, origin are known to possess so-called superantigenic properties (26, 27). Their particular properties of direct stimulation of T lymphocytes, specific to different antigens, by binding to the V.beta. region of certain "T" receptors, have suggested the hypothesis that such molecules are intimately associated with the etiopathogenic process of MS (28). Now, one of the characteristic effects of these superantigens on T cells is the premature induction, under certain conditions, of a programmed cell death or apoptosis (29). Superantigens also have properties of binding to HLA class II molecules at the surface of cells presenting the antigen (27). It may thus be recalled that astrocytes possess the capacity to express HLA class II antigens at their plasma membrane, and in particular in response to certain pro-inflammatory cytokines such as gamma interferon or tumor necrosis factor alpha (TNF-alpha) (30). However, no superantigen has yet been demonstrated in multiple sclerosis.
Moreover, many "untimely" apoptotic processes, as opposed to the normal apoptotic processes linked, for example, to the development of the nervous system (31), may take place in the absence of superantigen, either in the context of viral infection (32) or in the context of stimulation of a cell receptor (33). It is also of interest to note that an apoptosis may be induced by the in vitro stimulation of a TNF-alpha membrane receptor (31), but such a phenomenon has not been studied in the nervous system, and a fortiori with astrocytes.
Some cytokines may hence trigger a pathogenic process and be produced, in particular by macrophages, and they have a cytotoxic effect on oligodendrocytes (34). It should be noted that TNF-alpha, as well as interleukin-1-alpha, interleukin-1-beta, interleukin-2, interleukin-6 and gamma interferon, do not in principle have a cytolytic effect on astrocytes, but induce, rather, an astrocyte proliferation (35). However, astrocytes can themselves be subjected to stimulations at the origin of a secretion of TNF-alpha. This may be observed, for example, in response to lipopolysaccharide bacterial toxins or to calcium ionophores (36). Thus, an involvement of the oligodendrocytes, and hence a destruction of the myelin layers, may take place indirectly via the TNF-alpha produced by astrocytes. Any molecule which induces such a cytokine production by astrocytes, irrespective of its specific effect on the latter (proliferation, differentiation or cytopathogenic effect) is hence potentially demyelinating. TNF-alpha induces an oligodendrocytic cytotoxic mechanism whose primary effects are marked by a swelling of the myelin structures, suggestive of an ion channel-mediated action (34). A correlation seems, moreover, to exist between the production of TNF-alpha in vivo and exacerbations of MS (37). The role of astrocytes in such a production of TNF-alpha in patients suffering from MS is, however, unknown.
At all events, since astrocytes are capable of producing TNF-alpha and of copresenting a target antigen with HLA class II antigens to immunocompetent cells, which are themselves recruited by a cytopathic and/or inflammatory process, they are, in fact, at the pivotal point of the immunopathological interactions such as may be observed in the demyelinating lesion process which characterizes MS.
Another type of molecule capable of playing a part in the pathogenic process leading to the formation of a demyelination plaque, and then to an astrocytic gliosis, consists of the so-called heat shock proteins (HSP) or stress proteins. These proteins constitute a relatively conserved phylogenetic family and are to be found both in prokaryotes and in higher vertebrates (38). Their synthesis is inducible in eukaryotic cells by various stresses, in particular infectious or thermal stresses, and their interspecies antigenic likeness has suggested a possible induction of autoimmunity in man by infectious bacteria carrying HSP, such as Mycobacterium tuberculosis (39).
Bacterial antigens capable of possessing superantigenic properties or of being heat shock proteins have been implicated in the induction of exacerbations of MS, without it being clearly established to date whether they are chance cofactors of pathogenicity or are etiological agents, or are alternatively pathogens sharing common properties responsible for an identical pathogenicity (40, 42).
This type of mechanism involves, on the one hand, the presence of exogenous HSP (for example bacterial HSP) against which the immunocompetent cells become sensitized, and on the other hand the expression of endogenous HSP (of the infected body) at the surface of cells expressing HLA class II antigens. If the cellular HSP share common epitopes with the exogenous HSP, they may be recognized as "infectious" by sensitized lymphocytes. Their role has also been mentioned in MS (42). The "gliotoxicity" of such molecules may proceed via the immune response and, possibly, via the astrocytic cells or the microgliocytes (other macrophage glial cells capable of presenting a specific antigen), but the distinctive cytopathic effects of some HSP on glial cells are unknown in view of the absence of exhaustive studies in this field.
Some investigations carried out have provided arguments in favor of a viral etiology of MS (in particular 43, 44, 45 and WO-93/20188, the content of which is incorporated by way of reference).
Following the abovementioned investigations, the present inventors were led to look for one or more factors which are effectors of the pathogenic process ending in the typical formation of demyelination plaques and in an astrocytic gliosis.
Although cultures of multiple sclerosis blood monocytes/macrophages contributed to supporting a viral hypothesis during the abovementioned investigations, other aspects of the role of macrophage cells in the pathogenesis of this disease, which do not necessarily involve a viral agent, must be taken into consideration; in this connection, a hypothesis has hence been put forward and verified, according to which one or more factor(s) which is/are toxic to macroglial cells (astrocytes, oligodendrocytes) might be produced by the monocytes of patients suffering from MS.
Although the molecular factors in question are unknown, a toxic factor originating either from immune or inflammatory cells or from a viral or bacterial agent, or alternatively induced by these latter in the surrounding cells, is capable of initiating a process ending in an acute or chronic inflammatory response.
The double uncertainty about the possible retroviral origin of MS (46) and about the possible contribution of a bacterial agent to its pathogenesis (40, 47) confirms the investigators in their search for factors which are cytotoxic to macroglial cells in patients suffering from MS. In effect, the glial cells are the ones which constitute the main target of the neuropathological process in MS.
A. N. Davison et al. (48) have studied the activity of oligodendrocytes, which are the cells involved in myelin synthesis, and in particular the factors capable of participating as a myelin synthesis inhibitor. These investigations are, however, exclusively limited to oligodendrocytes, and do not enable progress to be made in explaining the pathogenic process described above.